Cation-chloride cotransporters, e.g. Na-K-21 and K-CI cotransporters, play fundamental roles in a variety of cells and tissues. In the nervous system, they modulate inhibitory synaptic transmission and participate to the movement of salt and fluid across epithelia. The Na-K-2CI cotransporter, NKCC1, accumulates Cl- in sensory neurons (DRG, olfactory), thus promoting GABA depolarizing responses. The cotransporter also participates in the production of the inner ear fluid and the reabsorption of K+ from the CSF. Knockout of NKCC1 leads to multiple phenotypes including increased sensitivity to pain and sensorineural deafness. Cation-chloride cotransporters are regulated by a variety of stimuli, most of them (if not all of them) converging to phosphorylation/dephosphorylation of the transporters, but not much is known about the kinases affecting them. We have recently identified, as interactors of cation-chloride cotransporters, stress kinases related to the yeast Ste20 kinase family. Together with WNK4, a kinase that is associated with human hypertension, the stress kinases modulate the activity of the cotransporters. We have also identified a SPAK-interacting tyrosine kinase which negatively regulates NKCC1 activity. To understand the physiological significance of the interaction between these kinases and the cotransporters, we propose to 1) Define the role of the stress kinases and WNK4 in modulating the activity of NKCC1 and 2) Define the role of the tyrosine kinase AATYK in the regulation of NKCC1 activity. This will be achieved through functional studies of cotransporter and kinase mutants in Xenopus laevis oocytes and through in vitro phosphorylation experiments. We will also address the role of PP1 and its putative binding to a site that overlaps with a SPAK binding domain. 3) Utilize a cell line which expresses NKCC1, SPAK, OSR1, WNK4, and AATYK to address through silencing experiments the specific role of each kinase in the regulation of the cotransporter. These studies will elucidate novel aspects of cation-chloride cotransporter function and regulation and provide a better understanding of cation-chloride cotransporter links to CNS-related disorders. Mechanisms that transport inorganic ions across cell membranes are involved in a variety of disorders: salt wasting disorders, hypertension, nerve degeneration and brain hyperexcitability. The transporters do not function in isolation but are tightly regulated by a variety of other proteins that also have the potential to participate in these diseases. Detailed studies of the interaction between regulatory proteins and the transporters are of critical importance for understanding these human disorders.